Complementary Technologies and Advancements using CRISPR and LentiORFs-S2

RNAi screening has made it possible to identify new genes, or gene networks, that are involved in a wide vari¬ety of biological processes, including assays relevant to signal transduction, cell viability, cell morphology, protein localization and function, drug resistance, and responses of host cells to pathogens. As such, RNAi continues to help us gain critical insights into the mechanisms underlying human disease and accelerate the development of treatments for cancer and a host of other disorders. The intersection between RNAi screening and complementary approaches such as CRISPR-Cas9-mediated genome editing has opened up new opportunities for assay development, screening and validation. The successful implementation of genome-editing technologies in several species suggests this will serve as an important and relevant tool for validation studies in numerous cell lines and model systems. Additionally, RNAi rescue experiments using LentiORFs serve an important role in further validating and boosting confidence of screened hits. As we continue to develop new strategies to improve genome-wide RNAi screening and validation, the significance of RNAi as a research tool will remain for many years to come.

Currently the US EPA has not promulgated a method for the analysis of Total Nitrogen (TN). Most states that have TN regulations require permitee's to measure TN as the composite of TKN, Nitrate and Nitrite. This approach is costly, time consuming, and also susceptible to variability based on the methods used for the individual analytes Merck, KGaA, Darmstadt, Germany is excited to announce the introduction of a Total Nitrogen Test Method consisting of a single spectrophotometric test kit. The method has been evaluated in a multilab study and this webinar will present the results of that study as well as an overview of the savings made possible by utilizing this new method for testing Total Nitrogen.

In the continued effort to save you time and simplify your workflow, we’ve developed a Fontana-Masson Staining Kit with a protocol that is 30x faster than the standard protocol. In this webinar, the researcher who developed the kit explains how easy it is to use this new rapid microwave technique. If you need to stain melanin and argentaffin cells in tissue sections, take a few minutes to watch this webinar - and save yourself many more minutes in the future by using this kit.

In the continued effort to save you time and simplify your workflow, we’ve developed a Fontana-Masson Staining Kit with a protocol that is 30x faster than the standard protocol. In this webinar, the researcher who developed the kit explains how easy it is to use this new rapid microwave technique. If you need to stain melanin and argentaffin cells in tissue sections, take a few minutes to watch this webinar - and save yourself many more minutes in the future by using this kit.

Many Pharmacopeia materials contain water as hydrates or in adsorbed form. Therefore the determination of the water content is important to maintaining compliance with the Pharmacopeia guidelines. Generally one of the three methods are given in the monographs, Method I (Titrimetric), Method II (Azeotropic), or Method III (Gravimetric). This poster will discuss the application of these methods to moisture determinations focusing on Karl Fischer techniques.

The complexity and diversity of food matrices, and the need for clean extracts to be injected into analytical instrumentation may imply the use of tedious and time-consuming sample preparation strategies that often produce significant laboratory waste. The development of a new matrix-compatible solid phase microextraction (SPME) coating, namely PDMS/DVB/PDMS, bearing enhanced antifouling properties, enables the analysis of complex food matrices by direct immersion SPME, and helps to overcome issues related to extensive sample pre-treatment and instrumental contamination. This webinar will focus on recent advances made toward the analysis of contaminants in complex food samples by using the new matrix-compatible SPME coating, and will describe the optimization of important parameters to be considered when performing Direct Immersion SPME (DI-SPME).

Assorted case studies using MIFC for analysis of protein and vaccine formulations will be presented, with an emphasis on measurements and samples that pose challenges for current techniques- including detection of small and transparent particles, direct analysis of highly concentrated formulations, and fluorescence characterization of particle type, chemical composition, and heterogeneous interactions.

CRISPR-Cas9 nucleases have revolutionized genome editing enabling unprecedented efficiency of targeted mutagenesis. The ability to perform large-scale, whole genome loss-of-function screens has allowed for the rapid identification of gene pathways and targets relevant to drug resistance and disease. The CRISPR SAM activator libraries extend the reach of whole genome screening beyond simple knockout, delivering transcriptional co-regulators to a specific human or mouse target sequence using modified dCas9/gRNA complexes. In this webinar, we will introduce new strategies for forward genetic screening using CRISPR SAM activator libraries. We will discuss the application of this technology as it pertains to experimental design, delivery mechanisms, data analysis, and target validation.

Mass spectrometry (MS) is an accepted research tool for both academic and industrial laboratories. As MS continues to gain ground in clinical and industrial testing, the requirements for high throughput, high sensitivity and high accuracy analyses put more emphasis on sample preparation. Solid Phase Microextraction (SPME) is well suited for this purpose as it requires minimal sample, provides pre-concentration of analytes, and allows for quantitative determinations.
This webinar will focus on the use of microextraction devices for direct MS analysis for applications that do not require chromatographic separation. Such analysis often results in very high throughput and more immediate results in comparison to traditional methods. Several Direct MS interfaces will be reviewed. The relevant overview of the literature as applied to the direct MS analysis of microextracted samples will be presented. The presentation also will discuss in more detail the coupling of Direct Analysis Real Time (DART) with a new type of solid phase microextraction devices (known as BioSPME).

The development of human iPSC technology offers researchers the ability to more accurately generate physiologically relevant models of disease and normal tissues in the laboratory. Advances in iPSC generation have allowed many laboratories to make their own cell lines; however, researchers rarely have the resources needed to establish stocks, undertake quality control and share their own de novo iPSC cell lines with other laboratories. A pre-existing and established iPSC collection therefore allows iPSC researchers to obtain “off the shelf” access to a large, robust and reliable supply of iPS cell lines that represent diverse donor to donor variability and which include disease status normal controls and gene edited cell lines. iPSCs from ECACC are standardised and quality controlled and have the benefit of coming from a trusted and internationally recognised collection with worldwide distribution.

Various methods for the extraction and purification of recombinant proteins from bacteria are in use and have been well described. Common methods for cell lysis involve mechanical disaggregation of cells, such as sonication, but this can be time-consuming when working with multiple samples and more importantly can result in localized heating leading to loss of protein activity. In this webinar, we provide data showing that gentle detergent based lysis leads to greater protein activity and higher yields when compared to mechanical methods such as sonication. We also investigate the use of dual purification strategies and explore optimizing conditions to improve the purity of protein purification techniques.

The in vitro micronucleus assay is one of the most widely used tests to quantify genotoxicity and cytotoxicity, especially as a screening tool in the development of chemicals and pharmaceuticals. Micronuclei (MN) are formed from whole chromosomes or chromosome fragments that lag behind during the metaphase-anaphase transition and are excluded from the main nucleus following division. MN form into small, rounded bodies surrounded by their own nuclear envelope and represent chromosomal mutations that can be used as an endpoint in genotoxicity testing. Typically performed by manual microscopy, the MN assay is laborious and can be subject to scorer bias. To overcome this, automated microscopy and conventional flow cytometry methods have been developed. However, these methods suffer from several limitations such as the requirement to create high quality slides in the case of microscopy and the lack of visual confirmation of MN in the case of flow cytometry. The ImageStream®X (ISX) imaging flow cytometer has the potential to overcome these limitations as it combines the speed, statistical robustness and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery.
In this webinar, adaptation of the in vitro MN assay to an imaging flow cytometry-based method will be described. Using the ISX Mark II imaging flow cytometer, images of micronucleated mono- and binucleated cells as well as polynucleated cells can be captured at a high flow rate and automatically identified and scored in the Image Data Exploration and Analysis Software (IDEAS®) that accompanies the ISX. A data analysis template created specifically for this application allows for the determination of both genotoxicity and cytotoxicity following treatment with known clastogens and aneugens. This work is the first demonstration of fully automated method for performing the in vitro MN assay on an imaging flow cytometry platform.

This webinar will provide an overview of recent advances in RAFT agents (900150, 900157 and 900158). New dithiocarbamate RAFT agents are extremely versatile, RAFT agents with wide-spread monomer applicability. The RAFT agents have the distinct advantage of low odour levels and in addition to this and the derived polymers do not develop odour on storage as no low molar mass thiols are generated. In most cases they are an appropriate replacement for trithiocarbonate RAFT agents. The new RAFT agents have the ability to control polymerization of both MAMs (more activated monomers) and LAMs (less activated monomers) and have been shown to be suitable for the synthesis of poly(MAM)-block-poly(LAM), specifically poly(DMA)-blockpoly(VAc).

Mass spectrometry-based protein assays impart increased specificity and more rapid development times versus traditional methods, such as ELISA. Coupled with immunoaffinity enrichment, LC-MS/MS is becoming a powerful tool for the quantitation of proteins in plasma. Such methods typically rely on synthetic stable isotope labeled (SIL) peptide internal standards to correct for instrumental variability. For more accurate protein quantitation by LC-MS/MS, experimental variations throughout the entire sample preparation workflow, including protein fractionation, immunoaffinity enrichment, and enzymatic digestion, must be accounted for. An ideal way of improving assay reproducibility is to add a full-length stable isotope labeled recombinant protein, that is equivalent to the native target protein, to the sample at the initial stage of the assay workflow. We have developed a set of stable-isotope-labeled monoclonal antibodies expressed in CHO cells as well as SIL versions of several clinically-relevant human proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin (manufactured as a Certified Reference Material). We will present data to demonstrate that the use of full-length SIL proteins and antibodies as internal standards allows for more accurate and rapid quantitation of biotherapeutic antibodies and clinically-relevant human protein biomarkers in plasma by LC-MS/MS.

Mass spectrometry-based protein assays impart increased specificity and more rapid development times versus traditional methods, such as ELISA. Coupled with immunoaffinity enrichment, LC-MS/MS is becoming a powerful tool for the quantitation of proteins in plasma. Such methods typically rely on synthetic stable isotope labeled (SIL) peptide internal standards to correct for instrumental variability. For more accurate protein quantitation by LC-MS/MS, experimental variations throughout the entire sample preparation workflow, including protein fractionation, immunoaffinity enrichment, and enzymatic digestion, must be accounted for. An ideal way of improving assay reproducibility is to add a full-length stable isotope labeled recombinant protein, that is equivalent to the native target protein, to the sample at the initial stage of the assay workflow. We have developed a set of stable-isotope-labeled monoclonal antibodies expressed in CHO cells as well as SIL versions of several clinically-relevant human proteins expressed in E. coli, such as IGF1, and in mammalian HEK293 cells, such as Thyroglobulin (manufactured as a Certified Reference Material). We will present data to demonstrate that the use of full-length SIL proteins and antibodies as internal standards allows for more accurate and rapid quantitation of biotherapeutic antibodies and clinically-relevant human protein biomarkers in plasma by LC-MS/MS.

1. The kidney proximal tubule is the primary site of drug-induced nephrotoxicity. I will describe the development of a 3-dimensional flow-directed proximal tubule microphysiological system (MPS). The kidney MPS recapitulates the synthetic, metabolic and transport activities of kidney proximal tubule cells. This MPS is as an ideal platform for ex vivo modeling of nephrotoxicity. Towards this goal, we have evaluated nephrotoxicity in response to challenge with multiple toxicants, including the heavy metal pollutant cadmium, antisense oligonucleotides, the antibiotic polymyxin B and the Chinese herbal product aristolochic acid. We believe that MPS technologies will have major impacts on predictive toxicity testing and human risk assessment. Animal and in vitro systems do not always faithfully recapitulate drug and xenobiotic responses in the clinic or occupational/environmental exposures, respectively. MPS technologies will refine safety assessment and reduce our need for surrogate animal testing. An ultimate goal is to create integrated human MPS organ systems that could replace animal models.

2. Nortis has developed a technology that is used to recapitulate functional units of human organs in microfluidic devices (chips). Such organ models include vasculature, kidney, and liver models for toxicology studies, blood-brain barrier models for drug transport studies, and vascularized tumor microenvironment models for drug efficacy studies.

Solid phase microexatraction or SPME is a green method for extraction of analytes out of a sample. Since SPME is a non-exhaustive extraction technique, some analysts believe that SPME is not quantifiable. This presentation will provide basic information for developing a method to extract and quantify analytes using SPME. Examples will be given on the extraction and quantification of analytes out of various matrices, and SPME will be compared to other extraction techniques such as QuEChERS and SPE. In this webinar, we will discuss some new SPME technologies such as SPME-OC (over-coated) fibers and BioSPME that help to isolate and quantify analytes from interfering compounds in the matrix. Guidelines will be provided for enhancement of precision using SPME.

The benefits of three dimensional (3D) cell culture are widely appreciated. More cell-based technologies are now becoming available that enable researchers to preserve the native 3D structure of cells in vitro. These can be broadly divided into three areas: aggregate-based methods; hydrogels and extra-cellular matrices; and inert scaffold-based technologies. Each has strengths and weaknesses and there is no one technology that satisfies all applications. Tissues in the body are mostly composed of different cell types that are often highly organized in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Alternatively in tumours, cancer cells form aggregates and tissue masses composed of different cell types. Recreation of these types of architecture will significantly evolve 3D cell culture to a new level where real tissue-like structures can be generated in vitro.

This webinar will review the alternative approaches available to researchers and provide an overview of their capabilities and example applications. More sophisticated models are developing as 3D cell culture technology becomes established and accepted as a means of creating more physiologically relevant cell-based assays. Methods that are relatively straightforward to use and that recreate the organized structure of real tissues will become valuable research tools for use in discovery, validation studies, and modelling disease.

Key areas covered:
• 2D vs 3D cell culture debate
• Review of alternative approaches and the development of new technologies
• Challenges facing 3D culture methods, in terms of technologies available and methods used
• Showcase applications where 3D technology makes a difference
• Future perspective for 3D cell culture technology and further development

Does western blotting give you more trouble than expected? Do you feel like your precious samples are being wasted on bad westerns? Join us and find out how you can improve your western blots! In this seminar, you will learn general guidelines for performing and troubleshooting your westerns, such as:

Understanding the movements, modifications and interactions of proteins within a cell is key to unraveling the fundamental tenets of biology. However, the low-level expression of many proteins, combined with the transient nature of their interactions and movements, makes analyzing and understanding these processes quite difficult. Duolink® PLA, which is based on the principles of the proximity ligation assay (PLA), offers a solution to overcome these hurdles and to study the actions of endogenous proteins within cells and tissues. Combining the specificity of antibodies with the sensitivity afforded by rolling circle amplification, Duolink® PLA allows you to detect, visualize, and quantitate proteins and their interactions (even single events) where they happen within cells or tissue, all without overexpression or genetic manipulation. This seminar will cover the basic assay principle and advantages of the Duolink® PLA technology, and discuss recent applications and developments of the technology that make it an excellent tool to understand the fundamental mechanisms of biology, as well as disease states. Applications of Duolink® PLA include the investigation of cellular responses to varying stimuli, receptor dimerization and signalling cascades, post-translational modifications, and regulation of protein expression. New developments include use in flow cytometry and multiplexed detection.

The Life Science Business of Merck KGaA, Darmstadt, Germany Webinar Channel features scientific presentations from key specialists in analytical chemistry, biology, chemistry and life sciences on the practical and technical aspects of new developments and innovations, to help advance your research.